1. Field of the Invention
The present invention relates to the field of molecular biology, and more particularly, the detection of nucleotide polymorphisms including single nucleotide polymorphisms, and still more particularly nucleotide polymorphisms associated with osteoporosis.
2. Description of the Related Art
Two or three distinct phases of changes to bone mass occur over the life of an individual (see Riggs, West J. Med. 154:63-77, 1991). The first phase occurs in both men and women, and proceeds to attainment of a peak bone mass. This first phase is achieved through linear growth of the endochondral growth plates, and radial growth due to a rate of periosteal apposition. The second phase begins around age 30 for trabecular bone (flat bones such as the vertebrae and pelvis) and about age 40 for cortical bone (e.g., long bones found in the limbs) and continues to old age. This phase is characterized by slow bone loss, and occurs in both men and women. In women, a third phase of bone loss also occurs, most likely due to postmenopausal estrogen deficiencies. During this phase alone, women may lose an additional 10% of bone mass from the cortical bone and 25% from the trabecular compartment (see Riggs, supra).
Loss of bone mineral content can be caused by a wide variety of conditions including, for instance, osteoporosis, osteopenia, bone dysplasia and bone fracture, and may result in significant medical problems. For example, osteoporosis is a debilitating disease in humans characterized by marked decreases in skeletal bone mass and mineral density, structural deterioration of bone including degradation of bone microarchitecture and corresponding increases in bone fragility and susceptibility to fracture in afflicted individuals. Osteoporosis in humans is preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone), a condition found in approximately 25 million people in the United States. Another 7-8 million patients in the United States have been diagnosed with clinical osteoporosis (defined as bone mineral content greater than 2.5 standard deviations below that of mature young adult bone). Osteoporosis is one of the most expensive diseases for the health care system, costing tens of billions of dollars annually in the United States. In addition to health care-related costs, long-term residential care and lost working days add to the financial and social costs of this disease. Worldwide approximately 75 million people are at risk for osteoporosis. See, e.g., Eisman, 1999 Endocrine Rev. 20:788; Giguere et al., 2000 Clin. Genet. 57:161; Zmuda et al., 1999 Genetic Epidemiol. 16:356; Uitterlinden, 1999 European Calcified Tissue Society on the World Wide Web at ectsoc.orq/reviews/005uitt.htm.
The frequency of osteoporosis in the human population increases with age, and among Caucasians is predominant in women (who comprise 80% of the osteoporosis patient pool in the United States). The increased fragility and susceptibility to fracture of skeletal bone in the aged is aggravated by the greater risk of accidental falls in this population. More than 1.5 million osteoporosis-related bone fractures are reported in the United States each year. Fractured hips, wrists, and vertebrae are among the most common injuries associated with osteoporosis. Hip fractures in particular are extremely uncomfortable and expensive for the patient, and for women correlate with high rates of mortality and morbidity.
Although osteoporosis has been defined as an increase in the risk of fracture due to decreased bone mass, none of the presently available treatments for skeletal disorders can substantially increase the bone density of adults. There is a strong perception among all physicians that drugs are needed which could increase bone density in adults, particularly in the bones of the wrist, spinal column and hip that are at risk in osteopenia and osteoporosis.
Current strategies for the prevention of osteoporosis may offer some benefit to individuals but cannot ensure resolution of the disease. These strategies include moderating physical activity (particularly in weight-bearing activities) with the onset of advanced age, including adequate calcium in the diet, and avoiding consumption of products containing alcohol or tobacco. For patients presenting with clinical osteopenia or osteoporosis, all current therapeutic drugs and strategies are directed to reducing further loss of bone mass by inhibiting the process of bone absorption, a natural component of the bone remodeling process that occurs constitutively.
For example, estrogen is now being prescribed to retard bone loss. There is, however, some controversy over whether there is any long term benefit to patients and whether there is any effect at all on patients over 75 years old. Moreover, use of estrogen is believed to increase the risk of breast and endometrial cancer.
High doses of dietary calcium, with or without vitamin D has also been suggested for postmenopausal women. However, high doses of calcium can often have unpleasant gastrointestinal side effects, and serum and urinary calcium levels must be continuously monitored (see Khosla and Rigss, Mayo Clin. Proc. 70:978-982, 1995).
Other therapeutics which have been suggested include calcitonin, bisphosphonates, anabolic steroids and sodium fluoride. Such therapeutics however, have undesirable side effects (e.g., calcitonin and steroids may cause nausea and provoke an immune reaction, bisphosphonates and sodium fluoride may inhibit repair of fractures, even though bone density increases modestly) that may prevent their usage (see Khosla and Rigss, supra).
No currently practiced therapeutic strategy involves a drug that stimulates or enhances the growth of new bone mass. Further the present invention provides methods for determining whether someone is susceptible to osteoporosis. Further, the present invention provides other, related advantages.
Summary of the Invention It is an aspect of the present invention to provide a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a subject, the sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism at a position that corresponds to a non-coding region of SEQ ID NO:1 indicates an increased risk of altered bone mineral density. In one embodiment the invention provides a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1; and determining gender of the subject, wherein the presence of at least one gender-associated sclerostin gene region nucleotide polymorphism indicates an increased risk of altered bone mineral density. In another embodiment the invention provides a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism in the sample indicates an increased risk of altered bone mineral density, and wherein the polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is selected from position 4103, 17966, 18293, 58083, 74235 and 91068.
In another embodiment there is provided a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a female subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism in the sample indicates an increased risk of decreased bone mineral density, and wherein the polymorphism is located at a nucleotide that corresponds to a GGA trinucleotide insertion between positions 10565 and 10566 in SEQ ID NO:1. In another embodiment the invention provides a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a male subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism in the sample indicates an increased risk of increased bone mineral density, and wherein the polymorphism is located at a nucleotide that corresponds to position 91068 in SEQ ID NO:1. In a further embodiment the polymorphism at position 91068 comprises an A91068G substitution.
Turning to another embodiment of the present invention, there is provided a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism in the sample indicates an increased risk of altered bone mineral density, and wherein the polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is not between positions 10354 and 16757.
In another embodiment the invention provides a method for determining a risk for or presence of altered bone mineral density in a subject, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism in a biological sample from a subject, said sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, wherein the presence of at least one sclerostin gene region nucleotide polymorphism in the sample indicates an increased risk of altered bone mineral density, and wherein the polymorphism is not located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is selected from the group consisting of position 10357 and a trinucleotide insertion between positions 10565 and 10566.
In still another embodiment the invention provides method for determining a risk for or presence of altered bone mineral density in a first subject suspected of having or being at risk for having altered bone mineral density, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism that is associated with altered bone mineral density in each of a first and a second biological sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, said first biological sample being obtained from said first subject and said second sample being obtained from a second subject known to be free of a risk or presence of altered bone mineral density, wherein the presence of at least one sclerostin gene region nucleotide polymorphism that is associated with altered bone mineral density in said first biological sample and the absence of said sclerostin gene region nucleotide polymorphism at a corresponding nucleotide in said second biological sample indicates an increased risk of altered bone mineral density, and wherein the polymorphism is not located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is selected from the group consisting of position 10357 and a trinucleotide insertion between positions 10565 and 10566.
According to another embodiment of the invention there is provided a method for determining a risk for or presence of altered bone mineral density in a first subject suspected of having or being at risk for having altered bone mineral density, comprising determining a presence or absence of at least one sclerostin gene region nucleotide polymorphism that is associated with altered bone mineral density in each of a first and a second biological sample comprising DNA having a sequence that corresponds to at least 50 consecutive nucleotides that are present in SEQ ID NO:1, said first biological sample being obtained from said first subject and said second sample being obtained from a second subject known to be free of a risk or presence of altered bone mineral density, wherein the presence of at least one sclerostin gene region nucleotide polymorphism that is associated with altered bone mineral density in said first biological sample and the absence of said sclerostin gene region nucleotide polymorphism at a corresponding nucleotide in said second biological sample indicates an increased risk of altered bone mineral density, and wherein the polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is not between positions 10354 and 16757. According to certain further embodiments of the above described methods, at least one sclerostin gene region nucleotide polymorphism is selected from a single nucleotide substitution at a nucleotide position in SEQ ID NO:1 that is selected from C4103G, C17966G, A18293G, T58083C, A74235G and A91068G
In certain other further embodiments of the above described methods, the presence in a sample from a female subject of at least one sclerostin gene region nucleotide polymorphism that is a GGA trinucleotide insertion between nucleotide positions 10565 and 10566 in SEQ ID NO:1 indicates an increased risk of decreased bone mineral density. In certain other further embodiments of the above described methods the step of determining comprises contacting at least one biological sample with at least one oligonucleotide primer having a nucleotide sequence that is complementary to a portion of a nucleic acid molecule having the sequence set forth in SEQ ID NO:1, under conditions and for a time sufficient to allow hybridization of said primer to the DNA. In certain further embodiments the method comprises detecting hybridization and extension of the oligonucleotide primer to produce a product, and therefrom determining the presence or absence of at least one sclerostin gene region nucleotide polymorphism. In certain other further embodiments of methods described above, the step of determining comprises contacting each of said first and second biological samples with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the DNA of said first sample and present in the DNA of said second sample, under conditions and for a time sufficient to allow hybridization of said primer to the DNA; and detecting hybridization and extension of the primer to the DNA of the first sample to produce a first product and hybridization and extension of the primer to the DNA of the second sample to produce a second product distinguishable from said first product, and therefrom determining the presence or absence of at least one sclerostin gene region nucleotide polymorphism. According to certain further embodiments the DNA in the sample is amplified. According to certain other further embodiments the DNA in the first sample is amplified and the DNA in the second sample is amplified. According to certain other further embodiments the oligonucleotide primer comprises a nucleic acid molecule which comprises a nucleotide sequence set forth in a sequence selected from SEQ ID NOS:2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 23, 24, 26, 27, 29 and 30.
Turning to another aspect, the present invention provides an isolated polynucleotide comprising a sclerostin gene region polymorphism, the polynucleotide comprising a nucleotide sequence selected from (a) the sequence set forth in SEQ ID NO:1 and containing at least one sclerostin gene region nucleotide polymorphism, wherein said polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is selected from position 4103, 10357, 10566, 17966, 18293, 58083, 74235 and 91068, (b) the sequence set forth in SEQ ID NO:1 and containing a single nucleotide substitution at a position corresponding to a nucleotide position in SEQ ID NO:1 that is selected from C4103G, C10357T, C17966G, A18293G, T58083C, A74235G and A91068G, (c) the sequence set forth in SEQ ID NO:1 and containing a GGA trinucleotide insertion between nucleotide positions 10565 and 10566 in SEQ ID NO:1, (d) the sequence set forth in any one of SEQ ID NOS:4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 32 and 33, and (e) a sequence that is fully complementary to any sequence of (a)-(d).
In another embodiment the invention provides an isolated polynucleotide of at least 25 nucleotides comprising a polynucleotide sequence that corresponds to a portion of the nucleic acid sequence set forth in SEQ ID NO:1 in which at least one sclerostin gene region nucleotide polymorphism is present, wherein the polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is not between positions 10354 and 16757. In a further embodiment the sclerostin gene region nucleotide polymorphism is selected from (a) a single nucleotide substitution at a position corresponding to a nucleotide position in SEQ ID NO:1 that is selected from C4103G, C10357T, C17966G, A18293G, T58083C, A74235G and A91068G, and (b) a GGA trinucleotide insertion at a position corresponding to a nucleotide position in SEQ ID NO:1 that is between nucleotide positions 10565 and 10566. In another embodiment then invention provides an isolated polynucleotide comprising a nucleotide sequence selected from SEQ ID NOS:2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 23, 24, 26, 27, 29 and 30. In certain embodiments the invention provides an immobilized polynucleotide, comprising any of the above described polynucleotides coupled to a solid support, and in certain other embodiments the invention provides a nucleic acid array comprising a plurality of such isolated nucleic acid molecules immobilized on a solid support. In another embodiment there is provided a kit for identifying a sclerostin gene region polymorphism, comprising any of the above-described isolated polynucleotides and an ancillary reagent.
In another embodiment the invention provides a method of stratifying human subjects according to sclerostin gene region polymorphisms, comprising determining presence or absence of at least one sclerostin gene region polymorphism in a biological sample obtained from each of a plurality of subjects, wherein (i) presence or absence of a sclerostin gene region polymorphism indicates altered bone mineral density, and (ii) the polymorphism is located at a nucleotide that corresponds to a nucleotide position of SEQ ID NO:1 that is not between positions 10354 and 16757, and therefrom stratifying said subjects. In a further embodiment the sclerostin gene region polymorphism is selected from (a) a single nucleotide substitution at a position corresponding to a nucleotide position in SEQ ID NO:1 that is selected from C4103G, C10357T, C17966G, A18293G, T58083C, A74235G and A91068G, and (b) a GGA trinucleotide insertion at a position corresponding to a nucleotide position in SEQ ID NO:1 that is between nucleotide positions 10565 and 10566. In certain further embodiments the method comprises determining gender of each subject, wherein the presence of at least one gender-associated sclerostin gene region nucleotide polymorphism indicates an increased risk of altered bone mineral density, and in certain still further embodiments the gender-associated sclerostin gene region nucleotide polymorphism is selected from (a) a GGA trinucleotide insertion at a position corresponding to a nucleotide position in SEQ ID NO:1 that is between nucleotide positions 10565 and 10566, presence of which in a female subject indicates an increased risk of decreased bone mineral density, and (b) an A-to-G substitution at a position corresponding to nucleotide position 91068 in SEQ ID NO:1, presence of which in a male subject indicates an increased risk of increased bone mineral density.
Accordingly, the present invention provides nucleotide sequences that are associated with polymorphisms in the SOST gene region. Sequences associated with these nucleotide polymorphisms include SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 32 and 33 as identified herein. As described in greater detail below, diseases and/or conditions associated with bone density can be detected and/or predicted by the methods of the current invention using nucleotide sequences disclosed herein. Exemplary diseases and/or conditions include without limitation, osteopenia, osteoporosis and gum diseases.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entireties as if fully set forth herein.